Use of at least one substrate of carboxylesterase and/or triacylglycerol lipase for detecting bacteria of the group Bacillus cereus
a technology of carboxylesterase and triacylglycerin, which is applied in the field of microbiological monitoring, can solve the problems of low specificity of substrates, chromogenic media generating false positives, and use of fluorogenic pc-plc substrates such as 4 mu-cp,
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example 1
[0112]1.1 Operating Procedure
[0113]For the purposes of this example, a lean base is used, the composition of which is detailed in table 1 presented below:
[0114]
TABLE 1composition of the lean baseCompoundsConcentration in g / LGlucose0.25Agar13Yeast extract2Sodium pyruvate0.25NaCl5Buffer0.16
[0115]The various compounds of this lean base are weighed one by one. The whole is taken up in a necessary volume of osmosis-treated water. The media are melted at 100° C. and sterilised by autoclaving (121° C. cycle for 15 minutes).
[0116]After the media have returned to 55° C., the 5-bromo-4-chloro-3-indoxyl-myristate (X-C14) and 5-bromo-4-chloro-3-indoxyl-palmitate (X-C16) enzyme substrates—both carboxylesterase and triacylglycerol-lipase substrates—are added. To do so, stock solutions are prepared at 75 g / L in a DMSO-type organic solvent. A volume of Tween 20 is added to the stock solution volume necessary to obtain a final concentration of enzyme substrates of 150 mg / L.
[0117]The media are divide...
example 2
[0132]2.1 Operating Procedure
[0133]In the autoclaved lean base in example 1 (the composition of which is presented in the aforementioned table 1), brought back to 55° C., different chromogenic or fluorogenic substrates were tested with different surface active agents (surfactants). 31 bacterial strains (of the genus Bacillus) were spot-test seeded, at 1 μL apiece from 0.5 MacFarland suspensions calibrated with the aid of a densitometer. The dishes were then incubated aerobically, for 24 hours, at 30-35° C.
[0134]The chromogenic enzyme substrates 5-bromo-4-chloro-3-indoxyl-myristate (X-C14) and 5-bromo-4-chloro-3-indoxyl-palmitate (X-C16) were used so as to obtain final enzyme substrate concentrations of 0.15 g / L, starting from 50 g / L stock solutions.
[0135]The following surfactants were used:[0136]Tween 20 6 g / L, and[0137]Triton X305: 3 and 6 g / L
[0138]2.2 Results
[0139]The results thus obtained are presented below, in Table 5.
[0140]
TABLE 5ResultsFertility ofFertility ofstrains of theSe...
example 3
[0144]3.1 Operating Procedure
[0145]In the autoclaved lean base in example 1 (the composition of which is presented in the aforementioned table 1), brought back to a temperature of 55° C., substrates of esterases (carboxylesterase) based on alizarin with carbon chains ranging from 12 to 16 carbon atoms were tested. The stock solutions of the substrates were produced at concentrations of 12.5, 25 and 50 g / L solubilised in DMSO-type organic solvent, mixed with a Tween-type surfactant and added to the media such that the end concentration of substrates was respectively 50, 100 and 200 mg / L, always with same quantity of solvent and surfactant added to the final media.
[0146]The bacterial strains were spot-test seeded, at 1 μL apiece from 0.5 MacFarland suspensions calibrated with the aid of a densitometer. The dishes were then incubated aerobically, for 24 hours, at 30-35° C.
[0147]3.2 Results
[0148]The results thus obtained are presented below, in table 6.
[0149]
TABLE 6ResultsFertility ofSe...
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